Protein glycosylation modification in methylotrophic yeast

ABSTRACT

The present invention provides genetically engineered strains of  Pichia  capable of producing proteins with reduced glycosylation. In particular, the genetically engineered strains of the present invention are capable of expressing either or both of an α-1,2-mannosidase and glucosidase II. The genetically engineered strains of the present invention can be further modified such that the OCH1 gene is disrupted. Methods of producing glycoproteins with reduced glycosylation using such genetically engineered stains of  Pichia  are also provided.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.12/424,924, filed Apr. 16, 2009, which is a continuation of U.S.application Ser. No. 10/672,484, filed Sep. 25, 2003, now U.S. Pat. No.8,354,268, which is a continuation of U.S. application Ser. No.09/896,594, filed Jun. 29, 2001, now U.S. Pat. No. 6,803,225, whichclaims the benefit of U.S. Provisional Application Ser. No. 60/215,676,filed Jun. 30, 2000.

FIELD OF THE INVENTION

The present invention relates to methods and vectors useful forgenetically modifying the glycosylation process in methylotrophic yeaststrains for the purpose of producing glycoproteins with reducedglycosylation. The present invention further relates to methylotrophicyeast strains generated using the present methods and vectors, as wellas glycoproteins produced from such genetically modified strains.

BACKGROUND OF THE INVENTION

The methylotrophic yeasts including Pichia pastoris have been widelyused for production of recombinant proteins of commercial or medicalimportance. However, production and medical applications of sometherapeutic glycoproteins can be hampered by the differences in theprotein-linked carbohydrate biosynthesis between these yeasts and thetarget organism such as a mammalian subject.

Protein N-glycosylation originates in the endoplasmic reticulum (ER),where an N-linked oligosaccharide (Glc₃Man₉GlcNAc₂) assembled ondolichol (a lipid carrier intermediate) is transferred to theappropriate Asn of a nascent protein. This is an event common to alleukaryotic N-linked glycoproteins. The three glucose residues and onespecific α-1,2-linked mannose residue are removed by specificglucosidases and an α-1,2-mannosidase in the ER, resulting in the coreoligosaccharide structure, Man₈GlcNAc₂. The protein with this core sugarstructure is transported to the Golgi apparatus where the sugar moietyundergoes various modifications. There are significant differences inthe modifications of the sugar chain in the Golgi apparatus betweenyeast and higher eukaryotes.

In mammalian cells, the modification of the sugar chain proceeds via 3different pathways depending on the protein moiety to which it is added.That is, (1) the core sugar chain does not change; (2) the core sugarchain is changed by adding the N-acetylglucosamine-1-phosphate moiety(GlcNAc-1-P) in UDP-N-acetyl glucosamine (UDP-GlcNAc) to the 6-positionof mannose in the core sugar chain, followed by removing the GlcNAcmoiety to form an acidic sugar chain in the glycoprotein; or (3) thecore sugar chain is first converted into Man₅GlcNAc₂ by removing 3mannose residues with mannosidase I; Man₅GlcNAc₂ is further modified byadding GlcNAc and removing 2 more mannose residues, followed bysequentially adding GlcNAc, galactose (Gal), and N-acetylneuraminic acid(also called sialic acid (NeuNAc)) to form various hybrid or complexsugar chains (R. Kornfeld and S. Kornfeld, Ann. Rev. Biochem. 54:631-664, 1985; Chiba et al J. Biol. Chem. 273: 26298-26304, 1998).

In yeast, the modification of the sugar chain in the Golgi involves aseries of additions of mannose residues by differentmannosyltransferases (“outer chain” glycosylation). The structure of theouter chain glycosylation is specific to the organisms, typically withmore than 50 mannose residues in S. cerevisiae, and most commonly withstructures smaller than Man₁₄GlcNAc₂ in Pichia pastoris. Thisyeast-specific outer chain glycosylation of the high mannose type isalso denoted hyperglycosylation.

Hyperglycosylation is often undesired since it leads to heterogeneity ofa recombinant protein product in both carbohydrate composition andmolecular weight, which may complicate the protein purification. Thespecific activity (units/weight) of hyperglycosylated enzymes may belowered by the increased portion of carbohydrate. In addition, the outerchain glycosylation is strongly immunogenic which is undesirable in atherapeutic application. Moreover, the large outer chain sugar can maskthe immunogenic determinants of a therapeutic protein. For example, theinfluenza neuraminidase (NA) expressed in P. pastoris is glycosylatedwith N-glycans containing up to 30-40 mannose residues. Thehyperglycosylated NA has a reduced immunogenicity in mice, as thevariable and immunodominant surface loops on top of the NA molecule aremasked by the N-glycans (Martinet et al. Eur J. Biochem. 247: 332-338,1997).

Therefore, it is desirable to genetically engineer methylotrophic yeaststrains in which glycosylation of proteins can be manipulated and fromwhich recombinant proteins can be produced that would not be compromisedin structure or function by large N-glycan side chains.

SUMMARY OF THE INVENTION

The present invention is directed to methods and vectors useful forgenetically modifying the glycosylation process in methylotrophic yeaststrains to produce glycoproteins with reduced glycosylation.Methylotrophic yeast strains generated using the present methods andvectors, as well as glycoproteins produced from such geneticallymodified strains are also provided.

In one embodiment, the present invention provides vectors useful formaking genetically engineered methylotrophic yeast strains which arecapable of producing glycoproteins with reduced glycosylation.

In one aspect, the present invention provides “knock-in” vectors whichare capable of expressing in a methylotrophic yeast strain one or moreproteins whose enzymatic activities lead to a reduction of glycosylationin glycoproteins produced by the methylotrophic yeast strain.

In a preferred embodiment, the knock-in vectors of the present inventioninclude a nucleotide sequence coding for an α-1,2-mannosidase or afunctional part thereof and are capable of expressing theα-1,2-mannosidase or the functional part in a methylotrophic yeaststrain. A preferred nucleotide sequence is a nucleotide sequenceencoding the α-1,2-mannosidase of a fungal species, and more preferably,Trichoderma reesei. Preferably, the α-1,2-mannosidase expression vectoris engineered such that the α-1,2-mannosidase or a functional partthereof expressed from the vector includes an ER-retention signal. Apreferred ER-retention signal is HDEL (SEQ ID NO: 1). Theα-1,2-mannosidase coding sequence can be operable linked to aconstitutive or inducible promoter, and a 3′ termination sequence. Thevectors can be integrative vectors or replicative vectors. Particularlypreferred α-1,2-mannosidase expression vectors include pGAPZMFManHDEL,pGAPZMFManMycHDEL, pPICZBMFManMycHDEL, pGAPZmManHDEL, pGAPZmMycManHDEL,pPIC9 mMycManHDEL and pGAPZmMycManHDEL.

In another preferred embodiment, the knock-in vectors of the presentinvention include a sequence coding for a glucosidase II or a functionalpart thereof and are capable of expressing the glucosidase II or thefunctional part in a methylotrophic yeast strain. A preferred nucleotidesequence is a nucleotide sequence encoding the glucosidase II of afungal species, and more preferably, Saccharomyces cerevisiae.Preferably, the glucosidase II expression vector is engineered such thatthe glucosidase II or a functional part thereof expressed from thevector includes an ER-retention signal. A preferred ER-retention signalis HDEL (SEQ ID NO: 1). The glucosidase II coding sequence can beoperable linked to a constitutive or inducible promoter, and a 3′termination sequence. The vectors can be integrative vectors orreplicative vectors. Particularly preferred glucosidase II expressionvectors include pGAPZAGLSII, pPICZAGLSII, pAOX2ZAGLSII, pYPTIZAGLSII,pGAPADEglsII, pPICADEglsII, pAOX2ADEglsII, pYPTIADEglsII,pGAPZAglsIIHDEL and pGAPADEglsIIHDEL.

Expression vectors which include both of an α-1,2-mannosidase expressionunit and a glucosidase II expression unit are also provided by thepresent invention.

In another aspect, the present invention provides “knock-out” vectorswhich, when introduced into a methylotrophic yeast strain, inactivate ordisrupt a gene thereby facilitating the reduction in the glycosylationof glycoproteins produced in the methylotrophic yeast strain.

In one embodiment, the present invention provides a “knock-out” vectorwhich, when introduced into a methylotrophic yeast strain, inactivatesor disrupts the Och1 gene. A preferred Och1 knock-out vector ispBLURA5′PpOCH1.

Still another embodiment of the present invention provides vectors whichinclude both a knock-in unit and a knock-out unit.

Furthermore, any of the knock-in or knock-out vectors of the presentinvention can also include a nucleotide sequence capable of expressing aheterologous protein of interest in a methylotrophic yeast.

Another embodiment of the present invention provides methods ofmodifying the glycosylation in a methylotrophic yeast by transformingthe yeast with one or more vectors of the present invention.

Strains of a methylotrophic yeast which can be modified using thepresent methods include, but are not limited to, yeast strains capableof growth on methanol such as yeasts of the genera Candida, Hansenula,Torulopsis, and Pichia. Preferred methylotrophic yeasts are of the genusPichia. Especially preferred are Pichia pastoris strains GS115 (NRRLY-15851), GS190 (NRRL Y-18014), PPF1 (NRRL Y-18017), PPY120H, yGC4, andstrains derived therefrom. Methylotrophic yeast strains which can bemodified using the present methods also include those methylotrophicyeast strains which have been engineered to express one or moreheterologous proteins of interest. The glycosylation on the heterologousproteins expressed from these previously genetically engineered strainscan be reduced by transforming such strains with one or more of thevectors of the present invention

Methylotrophic yeast strains which are modified by practicing thepresent methods are provided in another embodiment of the presentinvention.

A further aspect of the present invention is directed to methods ofproducing glycoproteins with a reduced glycosylation.

In accordance with such methods, a nucleotide sequence capable ofexpressing a glycoprotein can be introduced into a methylotrophic yeaststrain which has previously been transformed with one or more of thevectors of the present invention. Alternatively, a methylotrophic yeaststrain which has been genetically engineered to express a glycoproteincan be transformed with one or more of the vectors of the presentinvention. Moreover, if a methylotrophic yeast strain is not transformedwith a nucleotide sequence encoding a glycoprotein of interest or any ofthe vectors of the present invention, such yeast strain can betransformed, either consecutively or simultaneously, with both anucleotide sequence capable of expressing the glycoprotein and one ormore vectors of the present invention. Additionally, a methylotrophicyeast strain can be transformed with one or more of the present knock-inand/or knock-out vectors which also include a nucleotide sequencecapable of expressing a glycoprotein in the methylotrophic yeast strain.

Glycoproteins products produced by using the methods of the presentinvention, i.e., glycoproteins with reduced N-glycosylation, are alsopart of the present invention.

Kits which include one or more of the vectors of the present invention,or one or more strains modified to produce glycoproteins with reducedglycosylation, are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts vectors carrying an HDEL (SEQ ID NO: 1)-taggedTrichoderma reesei α-1,2-mannosidase expression cassette and describesthe way in which these vectors were constructed according to methodsknown in the art. Abbreviations used throughout construction schemes: 5′AOX1 or AOX1 P: Pichia pastoris AOX1 promoter sequence; Amp R:ampicillin resistance gene; ColE1: ColE1 origin of replication; 3′AOX1:3′ sequences of the Pichia pastoris AOX1 gene; HIS4: HIS4 gene of Pichiapastoris. AOX TT: transcription terminator sequence of the Pichiapastoris AOX1 gene; ORF: open reading frame; S: secretion signal; PTEF1: the promoter sequence of the Saccharomyces cerevisiaetranscription elongation factor 1 gene; P EM7: synthetic constitutiveprokaryotic promotor EM7; Zeocin: Zeocin resistance gene; CYC1 TT: 3′end of the S. cerevisiae CYC1 gene; GAP: promoter sequence of the Pichiapastoris glyceraldehyde-3-phosphate dehydrogenase gene; PpURA3: Pichiapastoris URA3 gene. As can be seen in this figure, the Trichodermareesei α-1,2-mannosidase was operably linked to the coding sequence forthe S. cerevisiae α-mating factor secretion signal sequence and furtheroperably linked at the 3′ terminus of the coding sequence to the codingsequence for an HDEL (SEQ ID NO: 1) peptide. The whole fusion constructwas operably linked to either the P. pastoris AOX1 promoter (inpPIC9MFManHDEL) or to the P. pastoris GAP promotor (in pGAPZMFManHDEL).

FIG. 2 depicts vectors carrying an HDEL (SEQ ID NO: 1)-tagged Musmusculus α-1,2-mannosidase IB expression cassette and describes the wayin which these vectors were constructed according to methods known inthe art. As can be seen in this figure, the catalytic domain of the Musmusculus α-1,2-mannosidase IB was operably linked to the coding sequencefor the S. cerevisiae α-mating factor secretion signal sequence andfurther operably linked at the 3′ terminus of the coding sequence to thecoding sequence for an HDEL (SEQ ID NO: 1) peptide. The whole fusionconstruct was operably linked to either the P. pastoris AOX1 promoter(in pPIC9 mManHDEL) or to the P. pastoris GAP promotor (inpGAPZmManHDEL). Furthermore, variants of the expression cassette weremade in which the coding sequence for a cMyc epitope tag was insertedbetween the coding sequence for the S. cerevisiae α-Mating Factorsecretion signal sequence and the coding sequence for the catalyticdomain of the Mus musculus α-1,2-mannosidase IB. This expressioncassette was also operably linked to either the P. pastoris AOX1promoter (in pPIC9 mMycManHDEL) or to the P. pastoris GAP promotor (inpGAPZmMycManHDEL).

FIG. 3 depicts vectors carrying a MycHDEL tagged Trichoderma reeseiα-1,2-mannosidase and the way in which these vectors were obtained. Theresulting fusion construction was again operably linked to either the P.pastoris AOX1 promoter (in pPICZBMFManMycHDEL) or to the P. pastoris GAPpromotor (in pGAPZMFManMycHDEL).

FIG. 4 demonstrates the intracellular localization of the MycHDEL-taggedTrichoderma reesei α-1,2-mannosidase and indicates ER-targeting byimmunofluorescence analysis. Panel A Western blotting. Yeast strainswere grown in 10 ml YPG cultures to an OD₆₀₀=10, diluted fivefold andgrown in YPM for 48 h. 1/50th of the culture medium and 1/65th of thecells were analysed by SDS-PAGE and Western blotting with the mousemonoclonal 9E10 anti-Myc antibody. The position of molecular weightmarker proteins are indicated with arrows. Lanes 1-5: cellular lysates.1,2: pGAPZMFManMycHDEL transformants. 3: non-transformed PPY12OH(negative control). 4,5: pPICZBMFManMycHDEL transformants. Lanes 6-10:culture media. 6: non transformed PPY12OH (negative control). 7,8:pGAPZMFManMycHDEL transformants. 9,10: pPICZBMFManMycHDEL transformants.Panel B Immunofluorescence microscopy. 1: phase contrast image of a P.pastoris cell (strain PPY12OH transformed with pGAPZMFManHDEL) at 1000×magnification. The nucleus is visible as an ellipse in the lower rightquadrant of the cell. 2: same cell as in 1, but in fluorescencemicroscopy mode to show localization of the T. reeseimannosidase-Myc-HDEL protein. The protein is mainly localized in acircular distribution around the nucleus (nuclear envelope), which istypical for an endoplasmic reticulum steady-state distribution. 3: phasecontrast image of a P. pastoris cell (strain PPY12OH transformed withpGAPZMFManHDEL) at 1000× magnification. 4: same cell in fluorescencemicroscopy to show localization of the Golgi marker protein OCH1-HA inP. pastoris strain PPY12OH. The dot-like distribution throughout thecytoplasm, with 3-4 dots per cell is typical for cis-Golgi distributionin P. pastoris.

FIG. 5 depicts the co-sedimentation of mannosidase-MycHDEL with ProteinDisulfide Isomerase in sucrose density gradient centrifugation. The toppanel shows the distribution over the different fractions of the sucrosegradient of the OCH1-HA Golgi marker protein. The middle panel showsthis distribution for the Protein Disulfide Isomerase endoplasmicreticulum marker protein. Finally, the bottom panel shows thedistribution of the MycHDEL-tagged Trichoderma reesei α-1,2-mannosidaseover the same fractions. It is concluded that the mannosidase-MycHDELalmost exactly matches the distribution of the ER marker PDI and thusmainly resides in the ER of the Pichia pastoris yeast cells.

FIG. 6 depicts the N-glycan analysis of Trypanosoma cruzitrans-sialidase coexpressed with Trichoderma reesei mannosidase-HDEL.Panel A: malto-oligosaccharide size reference ladder. Sizes of theglycans are expressed in Glucose Units (GU) by comparison of theirelectrophoretic mobility to the mobility of thesemalto-oligosaccharides. Panel B: N-glycans derived from recombinantTrypanosoma cruzi trans-sialidase expressed in Pichia pastoris. The peakat GU=9,2 corresponds to Man₈GlcNAc₂. Panel C: same analytes as panel 2,but after overnight treatment with 3 U/ml purified recombinant T. reeseiα-1,2-mannosidase. Panel D: N-glycans derived from recombinanttrans-sialidase co-expressed in Pichia pastoris with T. reeseimannosidase-HDEL (under control of the GAP promotor). The peak at GU=7,6corresponds to the Man₅GlcNAc₂ peak in the profile of RNase B (Panel F).Panel E: same analytes as panel D, but after overnight treatment with 3mU/ml purified recombinant T. reesei α-1,2-mannosidase. Panel F:N-glycans derived from bovine RNase B. These glycans consist ofMan₅GlcNAc₂ to Man₈GlcNAc₂. Different isomers are resolved, accountingfor the number of peaks for Man₇GlcNAc₂.

FIG. 7 depicts the processing of influenza haemagglutinin N-glycans byHDEL (SEQ ID NO: 1)-tagged Trichoderma reesei α-1,2-mannosidase and theHDEL (SEQ ID NO: 1)-tagged catalytic domain of murine α-1,2-mannosidaseIB. The Man₅GlcNAc₂ reference oligosaccharide runs at scan 1850 in thisanalysis (not shown). Panel 1: malto-oligosaccharide size referenceladder. Panel 2: N-glycans derived from recombinant influenzahaemagglutinin expressed in Pichia pastoris. The peak at scan 2250corresponds to Man₉GlcNAc₂ Panel 3: N-glycans derived from recombinanthaemagglutinin co-expressed in Pichia pastoris with T. reeseimannosidase-HDEL (under control of the GAP promotor). The peak at scan1950 corresponds to Man₆GlcNAc₂. Panel 4: Same analytes as for panel 3,but after overnight treatment with 30 mU purified recombinant T. reesei1,2-mannosidase. Panel 5: N-glycans derived from recombinanthaemagglutinin co-expressed in Pichia pastoris with mouse mannosidaseIB-HDEL (under control of the GAP promotor). Panel 6: same analytes asfor panel 5, but after overnight treatment with 30 mU purifiedrecombinant T. reesei α-1,2-mannosidase.

FIG. 8 graphically depicts vector pBLURA5′PpOCH1 and the way in which itwas constructed.

FIG. 9 depicts the scheme for disrupting the Pichia pastoris OCH1 geneby single homologous recombination using pBLURA5′PpOCH1.

FIG. 10 depicts the cell wall glycoprotein N-glycan analysis of theOch1-inactivated clone and three clones derived from thisOch1-inactivated clone by transformation with pGAPZMFManHDEL. Panel 1shows the analysis of a mixture of malto-oligosaccharides, the degree ofpolymerisation of which is given by the numbers on the very top of thefigure. This analysis serves as a size reference for the other panels.On the vertical axis of all panels, peak intensity in relativefluorescence units is given. Panel 2-6: analysis of the cell wallglycoprotein N-glycans of the following strains: Panel 2, non-engineeredP. pastoris strain yGC4; Panel 3, yGC4 transformed with pBLURA5′PpOch1;4-6, three clones of the strain of Panel 3, supplementarily transformedwith pGAPZMFManHDEL. Panel 7: the N-glycans derived from bovine RNaseB,consisting of a mixture of Man₅₋₉GlcNAc₂. As can be seen from comparisonbetween panel 2 and 3 and reference to panel 7, transformation withpBLURA5′PpOch1 leads to a strongly increased abundance of theMan₈GlcNAc₂ substrate N-glycan (named peak 1 in Panel 2) of OCH1p. Peak2 represents the Man₉GlcNAc₂ product of OCH1p. Furthermore, uponsupplementary transformation of pGAPZMFManHDEL, the major glycan on thecell wall glycoproteins of three independent clones is the Man₅GlcNAc₂end product (peak 3 in panel 4) of T. reesei α-1,2-mannosidase digestionof the Man₈GlcNAc₂ substrate.

FIG. 11 depicts the analysis of exactly the same glycan mixtures as inFIG. 10, but after an in vitro digest with 3 mU/ml purified Trichodermareesei α-1,2-mannosidase, overnight in 20 mM sodium acetate pH=5.0. Axisassignment is the same as in FIG. 10. More Man₅GlcNAc₂ is formed in thepBLURA5′PpOch1 transformed strain (Panel 3) than in the parent strain(Panel 2). Peaks in all panels before scan 3900 come from contaminantsand should be ignored in the analysis.

FIG. 12 depicts the expression vector pGAPZAGLSII (SEQ ID NO: 18). PTEF1: promotor of S. cerevisiae transcription elongation factor gene. PEm7: synthetic prokaryotic promotor. Zeocin: zeocine resistance markergene. CYC1 TT: transcription terminator of S. cerevisiae cytochrome C1gene. Col E1: bacterial origin of replication. GAP: promotor of the P.pasttoris GAP gene. GLS2: S. cerevisiae glucosidase II gene. AOX1 TT:transcription terminator of the P. pastoris AOX1 gene

FIG. 13 depicts the expression vector pAOX2ZAGLSII (SEQ ID NO: 16). PTEF1: promotor of S. cerevisiae transcription elongation factor gene. PEm7: synthetic prokaryotic promotor. Zeocin: zeocine resistance markergene. CYC1 TT: transcription terminator of S. cerevisiae cytochrome C1gene. Col E1: bacterial origin of replication. AOX2 P: promotor of theP. pastoris AOX2 gene. GLS2: S. cerevisiae glucosidase II gene. AOX1 TT:transcription terminator of the P. pastoris AOX1 gene

FIG. 14 depicts the expression vector pPICZAGLSII (SEQ ID NO: 20). PTEF1: promotor of S. cerevisiae transcription elongation factor gene. PEm7: synthetic prokaryotic promotor. Zeocin: zeocine resistance markergene. CYC1 TT: transcription terminator of S. cerevisiae cytochrome C1gene. Col E1: origin of replication. AOX1 P: promotor of the P. pastorisAOX1 gene. GLS2: S. cerevisiae glucosidase II gene. AOX1 TT:transcription terminator of the P. pastoris AOX1 gene

FIG. 15 depicts the expression vector pYPT1ZAGLSII (SEQ ID NO: 22). PTEF1: promotor of S. cerevisiae transcription elongation factor gene. PEm7: synthetic prokaryotic promotor. Zeocin: zeocine resistance markergene. CYC1 TT: transcription terminator of S. cerevisiae cytochrome C1gene. Col E1: origin of replication. P YPT1: promotor of the P. pastorisYPT1 gene. GLS2: S. cerevisiae glucosidase II gene. AOX1 TT:transcription terminator of the P. pastoris AOX1 gene.

FIG. 16 depicts the expression vector pGAPADE1glsII (SEQ ID NO: 19). AmpR: Ampillicin resistance marker gene. ADE1: P. pastoris ADE1 selectionmarker gene. GAP: promotor of the P. Pastoris GAP gene. GLS2: S.cerevisiae glucosidase II gene. AOX1 TT: transcription terminator of theP. pastoris AOX1 gene

FIG. 17 depicts the expression vector pAOX2ADE1glsII (SEQ ID NO: 17).Amp R: Ampillicin resistance marker gene. ADE1: P. pastoris ADE1selection marker gene. AOX2 P: promotor of the P. pastoris AOX2 gene.GLS2: S. cerevisiae glucosidase II gene. AOX1 TT: transcriptionterminator of the P. pastoris AOX1 gene.

FIG. 18 depicts the expression vector pPICADE1glsII (SEQ ID NO: 21). AmpR: Ampillicin resistance marker gene. ADE1: P. pastoris ADE1 selectionmarker gene. AOX1 P: promotor of the P. pastoris AOX1 gene. GLS2: S.cerevisiae glucosidase II gene. AOX1 TT: transcription terminator of theP. pastoris AOX1 gene.

FIG. 19 depicts the expression vector pYPT1ADE1glsII (SEQ ID NO: 23).Amp R: Ampillicin resistance marker gene. ADE1: P. pastoris ADE1selection marker gene. P YPT1: promotor of the P. pastoris YPT1 gene.GLS2: S. cerevisiae glucosidase II gene. AOX1 TT: transcriptionterminator of the P. pastoris AOX1 gene.

FIG. 20 depicts the expression vector pGAPZAglsIIHDEL (SEQ ID NO: 24).Amp R: Ampillicin resistance marker gene. ADE1: P. pastoris ADE1selection marker gene. GAP: promotor of the P. pastoris GAP gene. GLS2:S. cerevisiae glucosidase II gene. AOX1 TT: transcription terminator ofthe P. pastoris AOX1 gene.

FIG. 21 depicts the expression vector pGAPADE1glsIIHDEL (SEQ ID NO: 25).P TEF1: promotor of S. cerevisiae transcription elongation factor gene.P Em7: synthetic prokaryotic promotor. Zeocin: zeocine resistance markergene. CYC1 TT: transcription terminator of S. cerevisiae cytochrome C1gene. Col E1: bacterial origin of replication. GAP: promotor of the P.pastoris GAP gene. GLS2: S. cerevisiae glucosidase II gene. AOX1 TT:transcription terminator of the P. pastoris AOX1 gene.

FIG. 22 depicts the test of the GLSII activity assay using acommercially available yeast alpha-glucosidase (Sigma: Cat. No. G-5003).The assay mixture contains phosphate-citrate buffer pH 6.8, mannose,2-deoxy-D-glucose, the substrate4-methylumbellyferyl-alpha-D-glucopyranoside and alpha-glucosidase fromSigma. 1: assay mixture illuminated with UV-light after overnightincubation at 37° C.; 2: same as 1, but this time, the assay mixturelacks the alpha-glucosidase; 3: same as 1, but this time, the assaymixture lacks the substrate.

FIG. 23 depicts the results of the activity of recombinantly expressedGLSII from Pichia pastoris. All assay mixtures were incubated overnightat 37° C. and afterwards illuminated with UV-light. 1: assay with yeastalpha-glucosidase (Sigma: Cat. No. G-5003); 2: assay with the purifiedmedium of strain 18 (PPY12-OH transformed with pGAPZAGLSII); 3: assaywith purified medium of the WT PPY12-OH strain; 4: assay with thepurified medium of strain H3 (PPY12-OH transformed withpGAPZAglsIIHDEL).

DETAILED DESCRIPTION OF THE INVENTION

It has been established that the majority of N-glycans on glycoproteinsleaving the endoplasmic reticulum (ER) of Pichia have the coreMan₈GlcNAc₂ oligosaccharide structure. After the proteins aretransported from the ER to the Golgi apparatus, additional mannoseresidues are added to this core sugar moiety by differentmannosyltransferases, resulting in glycoproteins with large mannose sidechains. Such hyperglycosylation of recombinant glycoproteins isundesirable in many instances. Accordingly, the present inventionprovides methods and vectors for genetically modifying methylotrophicyeast strains to produce glycoproteins with reduced glycosylation.Methylotrophic yeast strains generated using the present methods andvectors, as well as glycoproteins produced from such geneticallymodified strains are also provided.

In one embodiment, the present invention provides vectors useful forgenetically modifying methylotrophic yeast strains to produceglycoproteins with reduced glycosylation.

In one aspect, the present invention provides “knock-in” vectors whichare capable of expressing in a methylotrophic yeast strain one or moreproteins whose enzymatic activities lead to a reduction of glycosylationin glycoproteins produced by the methylotrophic yeast strain. Accordingto the present invention, such proteins include, e.g., anα-1,2-mannosidase, a glucosidase II, or functional parts thereof.

In a preferred embodiment, the vectors of the present invention includea sequence coding for an α-1,2-mannosidase or a functional part thereofand are capable of expressing the α-1,2-mannosidase or the functionalpart in a methylotrophic yeast strain.

An α-1,2-mannosidase cleaves the α-1,2-linked mannose residues at thenon-reducing ends of Man₈GlcNAc₂, and converts this core oligosaccharideon glycoproteins to Man₅GlcNAc₂. In vitro, Man₅GlcNAc₂ is a very poorsubstrate for any Pichia Golgi mannosyltransferase, i.e., mannoseresidues can not be added to this sugar structure. On the other hand,Man₅GlcNAc₂ is the acceptor substrate for the mammalianN-acetylglucosaminyl-transferase I and is an intermediate for thehybrid- and complex-type sugar chains characteristic of mammalianglycoproteins. Thus, by way of introducing an α-1,2-mannosidase intomethylotrophic yeasts such as Pichia, glycoproteins with reduced mannosecontent can be produced.

According to the present invention, the nucleotide sequence encoding anα-1,2-mannosidase for use in the expression vector of the presentinvention can derive from any species. A number of α-1,2-mannosidasegenes have been cloned and are available to those skilled in the art,including mammalian genes encoding, e.g., a murine α-1,2-mannosidase(Herscovics et al. J. Biol. Chem. 269: 9864-9871, 1994), a rabbitα-1,2-mannosidase (Lal et al. J. Biol. Chem. 269: 9872-9881, 1994) or ahuman α-1,2-mannosidase (Tremblay et al. Glycobiology 8: 585-595, 1998),as well as fungal genes encoding, e.g., an Aspergillus α-1,2-mannosidase(msdS gene), a Trichoderma reesei α-1,2-mannosidase (Maras et al. J.Biotechnol. 77: 255-263, 2000), or a Saccharomyces cerevisiaeα-1,2-mannosidase. Protein sequence analysis has revealed a high degreeof conservation among the eukaryotic α-1,2-mannosidases identified sofar.

Preferably, the nucleotide sequence for use in the present vectorsencodes a fungal α-1,2-mannosidase, more preferably, a Trichodermareesei α-1,2-mannosidase, and more particularly, the Trichoderma reeseiα-1,2-mannosidase described by Maras et al. J. Biotechnol. 77: 255-63(2000).

According to the present invention, the nucleotide sequence can alsocode for only a functional part of an α-1,2-mannosidase.

By “functional part” is meant a polypeptide fragment of anα-1,2-mannosidase which substantially retains the enzymatic activity ofthe full-length protein. By “substantially” is meant at least about 40%,or preferably, at least 50% or more of the enzymatic activity of thefull-length α-1,2-mannosidase is retained. For example, as illustratedby the present invention, the catalytic domain of the murineα-1,2-mannosidase IB constitutes a “functional part” of the murineα-1,2-mannosidase IB. Those skilled in the art can readily identify andmake functional parts of an α-1,2-mannosidase using a combination oftechniques known in the art. Predictions of the portions of anα-1,2-mannosidase essential to or sufficient to confer the enzymaticactivity can be made based on analysis of the protein sequence. Theactivity of a portion of an α-1,2-mannosidase of interest, expressed andpurified from an appropriate expression system, can be verified using invitro or in vivo assays described hereinbelow.

In accordance with the present invention, an α-1,2-mannosidase or afunctional part thereof expressed in a methylotrophic yeast strainpreferably is targeted to a site in the secretory pathway whereMan₈GlcNAc₂ (the substrate of α-1,2-mannosidase) is already formed on aglycoprotein, but has not reached a Golgi glycosyltransferase whichelongates the sugar chain with additional mannose residues.

Accordingly, in a preferred embodiment of the present invention, theα-1,2-mannosidase expression vector is engineered as such that theα-1,2-mannosidase or a functional part thereof expressed from the vectorincludes an ER-retention signal.

“An ER retention signal” refers to a peptide sequence which directs aprotein having such peptide sequence to be transported to and retainedin the ER. Such ER retention sequences are often found in proteins thatreside and function in the ER.

Multiple choices of ER retention signals are available to those skilledin the art, e.g., the first 21 amino acid residues of the S. cerevisiaeER protein MNS1 (Martinet et al. Biotechnology Letters 20: 1171-1177,1998). A preferred ER retention signal for use in the present inventionis peptide HDEL (SEQ ID NO: 1). The HDEL (SEQ ID NO: 1) peptidesequence, found in the C-terminus of a number of yeast proteins, acts asa retention/retrieval signal for the ER (Pelham EMBO J. 7: 913-918,1988). Proteins with an HDEL (SEQ ID NO: 1) sequence are bound by amembrane-bound receptor (Erd2p) and then enter a retrograde transportpathway for return to the ER from the Golgi apparatus.

According to the present invention, an ER retention signal can be placedanywhere in the protein sequence of an α-1,2-mannosidase, but preferablyat the C-terminus of the α-1,2-mannosidase.

The α-1,2-mannosidase for use in the present invention can be furthermodified, e.g., by insertion of an epitope tag to which antibodies areavailable, such as Myc, HA, FLAG and His6 tags well-known in the art. Anepitope-tagged α-1,2-mannosidase can be conveniently purified, ormonitored for both expression and intracellular localization.

An ER retention signal and an epitope tag can be readily introduced intoa protein of interest by inserting nucleotide sequences coding for suchsignal or tag into the nucleotide sequence encoding the protein ofinterest, using any of the molecular biology techniques known in theart.

In another preferred embodiment, the vectors of the present inventioninclude a sequence coding for a glucosidase II or a functional partthereof and are capable of expressing the glucosidase II or thefunctional part in the methylotrophic yeast strain.

It has been established that the initial N-linked oligosaccharide(Glc₃Man₉GlcNAc₂), transferred in the ER onto a protein, is cleaved inthe ER by specific glucosidases to remove the glucose residues, and by amannosidase to remove one specific α-1,2-linked mannose. It has beenobserved by the present inventors that some recombinant proteinsexpressed in Pichia have residual glucose residues on the sugar moietywhen such proteins leave the ER for the Golgi apparatus. The residualglucose molecules present on the sugar structure prevent the completedigestion of the sugar moiety by an α-1,2-mannosidase, and theintroduction of an exogenous glucosidase can facilitate the removal ofthese glucose residues.

According to the present invention, the nucleotide sequence encoding aglucosidase II can derive from any species. Glucosidase II genes havebeen cloned from a number of mammalian species including rat, mouse, pigand human. The glucosidase II protein from these mammalian speciesconsists of an alpha and a beta subunit. The alpha subunit is about 110kDa and contains the catalytic activity of the enzyme, while the betasubunit has a C-terminal HDEL (SEQ ID NO: 1) ER-retention sequence andis believed to be important for the ER localization of the enzyme. Theglucosidase II gene from S. cerevisiae has also been cloned (ORFYBR229c, located on chromosome II). This gene encodes a protein of about110 kDa, which shows a high degree of homology to the mammalian alphasubunits.

A preferred glucosidase II gene for use in the present vectors is from afungal species such as Pichia pastoris and S. cerevisiae. An example ofa fungal glucosidase II gene is the S. cerevisiae glucosidase II alphasubunit gene.

According to the present invention, the nucleotide sequence can alsoencode only a functional part of a glucosidase II. By “functional part”is meant a polypeptide fragment of a glucosidase II which substantiallyretains the enzymatic activity of the full-length protein. By“substantially” is meant at least about 40%, or preferably, at least 50%or more of the enzymatic activity of the full-length glucosidase II isretained. Functional parts of a glucosidase II can be identified andmade by those skilled in the art using a variety of techniques known inthe art.

In a preferred embodiment of the present invention, the glucosidase IIprotein is engineered to include an ER retention signal such that theprotein expressed in a methylotrophic yeast strain is targeted to the ERand retains therein for function. ER retention signals are as describedhereinabove, e.g., the HDEL (SEQ ID NO: 1) peptide sequence.

The glucosidase II for use in the present invention can be furthermodified, e.g., by insertion of an epitope tag to which antibodies areavailable, such as Myc, HA, FLAG, and His6 tag, which are well-known inthe art.

According to the present invention, the “knock-in” vectors can includeeither or both of an α-1,2-mannosidase coding sequence and a glucosidaseII coding sequence.

Further according to the present invention, the nucleotide sequencecoding for the enzyme to be expressed (e.g., an α-1,2-mannosidase or afunctional part thereof, or a glucosidase II or a functional partthereof) can be placed in an operable linkage to a promoter and a 3′termination sequence.

Promoters appropriate for expression of a protein in a methylotrophicyeast can include both constitutive promoters and inducible promoters.Constitutive promoters include e.g., the Pichia pastorisglyceraldehyde-3-phosphate dehydrogenase promoter (“the GAP promoter”).Examples of inducible promoters include, e.g., the Pichia pastorisalcohol oxidase I promoter (“the AOXI promoter”) (U.S. Pat. No.4,855,231), or the Pichia pastoris formaldehyde dehydrogenase promoter(“the FLD promoter”) (Shen et al. Gene 216: 93-102, 1998).

3′ termination sequences are sequences 3′ to the stop codon of astructural gene which function to stabilize the mRNA transcriptionproduct of the gene to which the sequence is operably linked, such assequences which elicit polyadenylation. 3′ termination sequences can beobtained from Pichia or other methylotrophic yeast. Examples of Pichiapastoris 3′ termination sequences useful for the practice of the presentinvention include termination sequences from the AOX1 gene, p40 gene,HIS4 gene and FLD1 gene.

The vectors of the present invention preferably contain a selectablemarker gene. The selectable marker may be any gene which confers aselectable phenotype upon a methylotrophic yeast strain and allowstransformed cells to be identified and selected from untransformedcells. The selectable marker system may include an auxotrophic mutantmethylotrophic yeast strain and a wild type gene which complements thehost's defect. Examples of such systems include the Saccharomycescerevisiae or Pichia pastoris HIS4 gene which may be used to complementhis4 Pichia strains, or the S. cerevisiae or Pichia pastoris ARG4 genewhich may be used to complement Pichia pastoris arg mutants. Otherselectable marker genes which function in Pichia pastoris include theZeo^(R) gene, the G418^(R) gene, and the like.

The vectors of the present invention can also include an autonomousreplication sequence (ARS). For example, U.S. Pat. No. 4,837,148describes autonomous replication sequences which provide a suitablemeans for maintaining plasmids in Pichia pastoris. The disclosure ofU.S. Pat. No. 4,837,148 is incorporated herein by reference.

The vectors can also contain selectable marker genes which function inbacteria, as well as sequences responsible for replication andextrachromosomal maintenance in bacteria. Examples of bacterialselectable marker genes include ampicillin resistance (Amp^(r)),tetracycline resistance (Tet^(r)), neomycin resistance, hygromycinresistance, and zeocin resistance (Zeo^(R)) genes.

According to the present invention, the nucleotide sequence encoding theprotein to be expressed in a methylotrophic yeast can be placed in anintegrative vector or a replicative vector (such as a replicatingcircular plasmid).

Integrative vectors are disclosed, e.g., in U.S. Pat. No. 4,882,279which is incorporated herein by reference. Integrative vectors generallyinclude a serially arranged sequence of at least a first insertable DNAfragment, a selectable marker gene, and a second insertable DNAfragment. The first and second insertable DNA fragments are each about200 nucleotides in length and have nucleotide sequences which arehomologous to portions of the genomic DNA of the species to betransformed. A nucleotide sequence containing a structural gene ofinterest for expression is inserted in this vector between the first andsecond insertable DNA fragments whether before or after the marker gene.Integrative vectors can be linearized prior to yeast transformation tofacilitate the integration of the nucleotide sequence of interest intothe host cell genome.

Replicative and integrative vectors carrying either or both of anα-1,2-mannosidase coding sequence or a glucosidase II coding sequencecan be constructed by standard techniques known to one of ordinary skillin the art and found, for example, in Sambrook et al. (1989) inMolecular Cloning: A Laboratory Manual, or any of a myriad of laboratorymanuals on recombinant DNA technology that are widely available.

Preferred vectors of the present invention carrying an α-1,2-mannosidaseexpression sequence include pGAPZMFManHDEL, pGAPZMFManMycHDEL,pPICZBMFManMycHDEL, pGAPZmManHDEL, pGAPZmMycManHDEL, pPIC9 mMycManHDELand pGAPZmMycManHDEL, which are further described in the Exampleshereinbelow.

Preferred vectors of the present invention carrying a glucosidase IIexpression sequence include pGAPZAGLSII, pPICZAGLSII, pAOX2ZAGLSII,pYPTIZAGLSII, pGAPADE1glsII, pPICADE1glsII, pAOX2ADE1glsII,pYPTIADE1glsII, pGAP

ZAglsIIHDEL and pGAPADE1glsIIHDEL, which are further described in theExamples hereinbelow.

In another aspect, the present invention provides “knock-out” vectorswhich, when introduced into a methylotrophic yeast strain, inactivate ordisrupt a gene thereby facilitating the reduction in the glycosylationof glycoproteins produced in the methylotrophic yeast strain.

In one embodiment, the present invention provides a “knock-out” vectorwhich, when introduced into a methylotrophic yeast strain, inactivatesor disrupts the Och1 gene.

The S. cerevisiae OCH1 gene has been cloned (Nakayama et al. EMBO J. 11:2511-2519, 1992). It encodes a membrane bound α-1,6-mannosyltransferase,localized in the early Golgi complex, that is functional in theinitiation of α-1,6-polymannose outer chain addition to the N-linkedcore oligosaccharide (Man₅GlcNAc₂ and Man₈GlcNAc₂) (Nakanishi-Shindo etal. J. Biol. Chem. 268: 26338-26345, 1993).

A Pichia sequence has been described in Japanese Patent Application No.07145005 that encodes a protein highly homologous to the S. cerevisiaeOCH1. For purpose of the present invention, this sequence is denotedherein as “the Pichia OCH1 gene”. Those skilled in the art can isolatethe OCH1 genes from other methylotrophic yeasts using techniques wellknown in the art.

According to the present invention, a disruption in the OCH1 gene of amethylotrophic yeast can result in either the production of an inactiveprotein product or no product. The disruption may take the form of aninsertion of a heterologous DNA sequence into the coding sequence and/orthe deletion of some or all of the coding sequence. Gene disruptions canbe generated by homologous recombination essentially as described byRothstein (in Methods in Enzymology, Wu et al., eds., vol 101:202-211,1983).

To disrupt the Och1 gene by homologous recombination, an Och1 knock-outvector can be constructed in such a way to include a selectable markergene. The selectable marker gene is operably linked, at both 5′ and 3′end, to portions of the Och1 gene of sufficient length to mediatehomologous recombination. The selectable marker can be one of any numberof genes which either complement host cell auxotrophy or provideantibiotic resistance, including URA3, LEU2 and HIS3 genes. Othersuitable selectable markers include the CAT gene, which conferschloramphenicol resistance on yeast cells, or the lacZ gene, whichresults in blue colonies due to the expression of activeβ-galactosidase. Linearized DNA fragments of an Och1 knock-out vectorare then introduced into host methylotrophic yeast cells using methodswell known in the art. Integration of the linear fragments into thegenome and the disruption of the Och1 gene can be determined based onthe selection marker and can be verified by, for example, Southern Blotanalysis.

Alternatively, an Och1 knock-out vector can be constructed in such a wayto include a portion of the Och1 gene to be disrupted, which portion isdevoid of any Och1 promoter sequence and encodes none or an inactivefragment of the Och1 protein. By “an inactive fragment”, it is meant afragment of the Och1 protein which has, preferably, less than about 10%and most preferably, about 0% of the activity of the full-length OCH1protein. Such portion of the OCH1 gene is inserted in a vector in such away that no known promoter sequence is operably linked to the OCH1sequence, but that a stop codon and a transcription termination sequenceare operably linked to the portion of the Och1 gene. This vector can besubsequently linearized in the portion of the OCH1 sequence andtransformed into a methylotrophic yeast strain using any of the methodsknown in the art. By way of single homologous recombination, thislinearized vector is then integrated in the OCH1 gene. Two Och1sequences are produced in the chromosome as a result of the singlehomologous recombination. The first Och1 sequence is the portion of theOch1 gene from the vector, which is now under control of the OCH1promoter of the host methylotrophic yeast, yet cannot produce an activeOCH1 protein as such Och1 sequence codes for no or an inactive fragmentof the OCH1 protein, as described hereinabove. The second Och1 sequenceis a full OCH1 coding sequence, but is not operably linked to any knownpromoter sequence and thus, no active messenger is expected to be formedfor synthesis of an active OCH1 protein. Preferably, an inactivatingmutation is introduced in the OCH1 sequence, to the 5′ end of the siteof linearization of the vector and to the 3′ end of the translationinitiation codon of OCH1. By “inactivating mutation” it is meant amutation introducing a stop codon, a frameshift mutation or any othermutation causing a disruption of the reading frame. Such mutation can beintroduced into an Och1 sequence using any of the site directedmutagenesis methods known in the art. Such inactivating mutation ensuresthat no functional OCH1 protein can be formed even if there exist somepromoter sequences 5′ to the Och1 sequence in the knock-out vector.

A preferred Och1 knock-out vector of the present invention ispBLURA5′PpOCH1.

If desired, either or both of a mannosidase expression sequence and aglucosidase expression sequence can be carried on the same plasmid usedto disrupt the OCH1 gene to create a “knock-in-and-knock-out” vector.

Additionally, any of the above-described vectors can further include anucleotide sequence capable of expressing a glycoprotein of interest ina methylotrophic yeast strain.

Another aspect of the present invention is directed to methods ofmodifying methylotrophic yeast strains to reduce glycosylation onproteins produced by the methylotrophic yeast strains. In accordancewith the present methods, methylotrophic yeast strains are modified bytransforming into these yeast strains with one or more, i.e., at leastone, knock-in and/or knock-out vectors of the present invention asdescribed herein above.

Methylotrophic yeast strains which can be modified using the presentmethods include but are not limited to yeast capable of growth onmethanol such as yeasts of the genera Candida, Hansenula, Torulopsis,and Pichia. A list of species which are exemplary of this class ofyeasts can be found in C. Anthony (1982), The Biochemistry ofMethylotrophs, 269. Pichia pastoris, Pichia methanolica, Pichia anomola,Hansenula polymorpha and Candida boidinii are examples of methylotrophicyeasts useful in the practice of the present invention. Preferredmethylotrophic yeasts are of the genus Pichia. Especially preferred arePichia pastoris strains GS115 (NRRL Y-15851); GS 190 (NRRL Y-18014)disclosed in U.S. Pat. No. 4,818,700; PPF1 (NRRL Y-18017) disclosed inU.S. Pat. No. 4,812,405; PPY120H and yGC4; as well as strains derivedtherefrom.

Methylotrophic yeast strains which can be modified using the presentmethods also include those methylotrophic yeast strains which have beengenetically engineered to express one or more heterologous glycoproteinsof interest. The glycosylation on the heterologous glycoproteinsexpressed from these previously engineered strains can be reduced bytransforming such strains with one or more of the vectors of the presentinvention.

The vectors of the present invention can be introduced into the cells ofa methylotrophic yeast strain using known methods such as thespheroplast technique, described by Cregg et al. 1985, or the whole-celllithium chloride yeast transformation system, Ito et al. Agric. Biol.Chem. 48:341, modified for use in Pichia as described in EP 312,934.Other published methods useful for transformation of the plasmids orlinear vectors include U.S. Pat. No. 4,929,555; Hinnen et al. Proc. Nat.Acad. Sci. USA 75:1929 (1978); Ito et al. J. Bacteriol. 153:163 (1983);U.S. Pat. No. 4,879,231; Sreekrishna et al. Gene 59:115 (1987).Electroporation and PEG1000 whole cell transformation procedures mayalso be used. Cregg and Russel Methods in Molecular Biology: PichiaProtocols, Chapter 3, Humana Press, Totowa, N.J., pp. 27-39 (1998).

Transformed yeast cells can be selected by using appropriate techniquesincluding but not limited to culturing auxotrophic cells aftertransformation in the absence of the biochemical product required (dueto the cell's auxotrophy), selection for and detection of a newphenotype, or culturing in the presence of an antibiotic which is toxicto the yeast in the absence of a resistance gene contained in thetransformants. Transformants can also be selected and/or verified byintegration of the expression cassette into the genome, which can beassessed by e.g., Southern Blot or PCR analysis.

In one embodiment, a methylotrophic yeast strain is transformed with avector which includes a nucleotide sequence coding for anα-1,2-mannosidase or a functional part thereof. The nucleotide sequenceis capable of expressing the α-1,2-mannosidase or the functional part inthe methylotrophic yeast strain, and is, preferably, integrated into thegenome of the methylotrophic yeast strain.

The expression of an α-1,2-mannosidase introduced in a methylotrophicyeast strain can be verified both at the mRNA level, e.g., by NorthernBlot analysis, and at the protein level, e.g., by Western Blot analysis.The intracellular localization of the protein can be analyzed by using avariety of techniques, including subcellular fractionation andimmunofluorescence experiments. An ER localization of anα-1,2-mannosidase can be determined by co-sedimentation of this enzymewith a known ER resident protein (e.g., Protein Disulfide Isomerase) ina subcellular fractionation experiment. An ER localization can also bedetermined by an immunofluorescence staining pattern characteristic ofER resident proteins, typically a perinuclear staining pattern.

To confirm that an α-1,2-mannosidase or a functional part thereofexpressed in a methylotrophic yeast strain has the expectedmannose-trimming activity, both in vitro and in vivo assays can beemployed. Typically, an in vitro assay involves digestion of an in vitrosynthesized substrate, e.g., Man₈GlcNAc₂, with the enzyme expressed andpurified from a methylotrophic yeast strain, and assessing the abilityof such enzyme to trim Man₈GlcNAc₂ to, e.g., Man₅GlcNAc₂. In in vivoassays, the α-1,2-mannosidase or a part thereof is co-expressed in amethylotrophic yeast with a glycoprotein known to be glycosylated withN-glycans bearing terminal α-1,2-linked mannose residues in such yeast.The enzymatic activity of such an α-1,2-mannosidase or a part thereofcan be measured based on the reduction of the number of α-1,2-linkedmannose residues in the structures of the N-glycans of the glycoprotein.In both in vitro and in vivo assays, the composition of a carbohydrategroup can be determined using techniques that are well known in the artand are illustrated in the Examples hereinbelow.

In another embodiment, a methylotrophic yeast strain is transformed witha vector which includes a nucleotide sequence coding for a glucosidaseII or a functional part thereof. The nucleotide sequence is capable ofexpressing the glucosidase II or the functional part in themethylotrophic yeast strain, and is, preferably, integrated into thegenome of the methylotrophic yeast strain.

The enzymatic activity of a glucosidase II or a functional part thereofexpressed in a transformed methylotrophic yeast strain can be assessedusing a variety of assays. For example, methylotrophic yeast cellstransformed with a sequence encoding a glucosidase II or a part thereofcan be set to grow on solid medium containing a substrate of theglucosidase, e.g., 5-bromo-4-chloro-3-indolyl-α-D-glucopyranoside or4-MU-α-D-Glc. When the enzyme is expressed by the Pichia and secretedextracellularly, the substrate is acted upon by the enzyme, giving riseto detectable signals around the colonies such as blue color orfluorescent glow. Alternatively, liquid culture medium containing theexpressed protein molecules can be collected and incubated in test tubeswith a substrate, e.g., p-nitrophenyl-α-D-glucopyranoside. The enzymaticactivity can be determined by measuring the specific product released.Moreover, in vivo assays can be employed, where a glucosidase II isco-expressed in yeast with a glycoprotein known to be N-glycosylatedwith glucose residues, e.g., influenza neuraminidase. The enzymaticactivity of the glucosidase II can be measured based on the reduction ofthe glucose content in the sugar chain(s) of the glycoprotein.

In still another embodiment of the present invention, a methylotrophicyeast strain is transformed with an Och1 knock-out vector. As a resultof the transformation and integration of the vector, the genomic Och1gene in the yeast strains is disrupted.

In a further embodiment of the present invention, a methylotrophic yeaststrain is transformed with any combination of an α-1,2-mannosidaseexpression vector, a glucosidase II expression vector, and an Och1knock-out vector. Such modification can be achieved by serial,consecutive transformations, i.e., introducing one vector at a time, oralternatively by co-transformation, i.e., introducing the vectorssimultaneously.

The modified methylotrophic yeast strains described herein above can befurther modified if desired. For example, additional disruption of genesencoding any other Pichia mannosyltransferases can be made. Genesencoding mammalian enzymes can also be introduced to produceglycoproteins having hybrid- or complex-type N-glycans, if desired.

Methylotrophic yeast strains which are modified by using the presentmethods, i.e., by transforming with one or more of the vectors of thepresent invention, form another embodiment of the present invention.

It should be understood that certain aspects of the present invention,especially the introduction of an intracellularly expressedα-1,2-mannosidase activity, are also useful to obtain a reducedglycosylation of the O-linked glycans on glycoproteins produced in amethylotrophic yeast, as it is known in the art that these O-linkedglycans consist mainly of α-1,2-linked mannose residues. O-linkedglycans as used herein refers to carbohydrate structures linked toserine or threonine residues of glycoproteins.

A further aspect of the invention is directed to methods of producing aglycoprotein with reduced glycosylation in a methylotrophic yeast,especially a glycoprotein heterologous to the methylotrophic yeast.

“A glycoprotein” as used herein refers to a protein which, inmethylotrophic yeasts, is either glycosylated on one or more asparaginesresidues or on one or more serine or threonine residues, or on bothasparagines and serine or threonine residues.

The term “reduced glycosylation” refers to a reduced size of thecarbohydrate moiety on the glycoprotein, particularly with fewer mannoseresidues, when the glycoprotein is expressed in a methylotrophic yeaststrain which has been modified in accordance with the present invention,as compared to a wild type, unmodified strain of the methylotrophicyeast.

In accordance with the present invention, the production of aglycoprotein of interest with reduced glycosylation can be achieved in anumber of ways. A nucleotide sequence capable of expressing aglycoprotein can be introduced into a methylotrophic yeast strain whichhas been previously modified in accordance with the present invention,i.e., a strain transformed with one or more of the vectors of thepresent invention and capable of producing glycoproteins with reducedglycosylation. Alternatively, a methylotrophic yeast strain which hasalready been genetically engineered to express a glycoprotein can betransformed with one or more of the vectors of the present invention.Otherwise, if a methylotrophic yeast strain does not express aglycoprotein of interest, nor is the strain transformed with any of thevectors of the present invention, such yeast strain can be transformed,either consecutively or simultaneously, with both a nucleotide sequencecapable of expressing the glycoprotein and one or more vectors of thepresent invention. Additionally, a methylotrophic yeast strain can betransformed with one or more of the present knock-in and/or knock-outvectors which also include a nucleotide sequence capable of expressing aglycoprotein in the methylotrophic yeast strain.

The nucleotide sequence capable of expressing a glycoprotein in amethylotrophic yeast can be made to include from 5′ to 3′, a promoter, asequence encoding the glycoprotein, and a 3′ termination sequence.Promoters and 3′ termination sequences which are suitable for expressionof a glycoprotein can include any of those promoters and 3′ terminationsequences described hereinabove.

The nucleotide sequence for expression of a glycoprotein can includeadditional sequences, e.g., signal sequences coding for transit peptideswhen secretion of a protein product is desired. Such sequences arewidely known, readily available and include Saccharomyces cerevisiaealpha mating factor prepro (αmf), Pichia pastoris acid phosphatase(PHO1) signal sequence and the like.

The nucleotide sequence for expression of a glycoprotein can be placedon a replicative vector or an integrative vector. The choice andconstruction of such vectors are as described hereinabove.

The nucleotide sequence capable of expressing a glycoprotein can becarried on the same replicative plasmid as a plasmid-borneα-1,2-mannosidase or glucosidase II expression unit. Alternatively, thenucleotide sequence containing the glycoprotein coding sequence iscarried on a separate plasmid or integrated into the host genome.

Glycoproteins produced can be purified by conventional methods.Purification protocols can be determined by the nature of the specificprotein to be purified. Such determination is within the ordinary levelof skill in the art. For example, the cell culture medium is separatedfrom the cells and the protein secreted from the cells can be isolatedfrom the medium by routine isolation techniques such as precipitation,immunoadsorption, fractionation or a variety of chromatographic methods.

Glycoproteins which can be produced by the methods of the presentinvention include, e.g., Bacillus amyloliquefaciens α-amylase, S.cerevisiae invertase, Trypanosoma cruzi trans-sialidase, HIV envelopeprotein, influenza virus A haemagglutinin, influenza neuraminidase,Bovine herpes virus type-1 glycoprotein D, human angiostatin, humanB7-1, B7-2 and B-7 receptor CTLA-4, human tissue factor, growth factors(e.g., platelet-derived growth factor), tissue plasminogen activator,plasminogen activator inhibitor-I, urokinase, human lysosomal proteinssuch as α-galactosidase, plasminogen, thrombin, factor XIII andimmunoglobulins. For additional useful glycoproteins which can beexpressed in the genetically engineered Pichia strains of the presentinvention, see Bretthauer and Castellino, Biotechnol. Appl. Biochem. 30:193-200 (1999), and Kukuruzinska et al. Ann Rev. Biochem. 56: 915-44(1987).

Glycoproteins produced by using the methods of the present invention,i.e., glycoproteins with reduced glycosylation, are also part of thepresent invention.

Still another aspect of the present invention provides kits whichcontain one or more of the knock-in vectors, knock-out vectors, orknock-in-and-knock-out vectors of the present invention described above.More particularly, a kit of the present invention contains a vectorcapable of expressing an α-mannosidase I in a methylotrophic yeast, avector capable of expressing a glucosidase II in a methylotrophic yeast,a vector capable of disrupting the Och1 gene in a methylotrophic yeast,a vector capable of expressing both a glucosidase II and anα-mannosidase, a vector a vector capable of disrupting the Och1 gene andcapable of expressing either or both of a glucosidase II and anα-mannosidase, or any combinations thereof.

The kit can also include a nucleotide sequence which encodes and iscapable of expressing a heterologous glycoprotein of interest. Suchnucleotide sequence can be provided in a separate vector or in the samevector which contains sequences for knocking-in or knocking out asdescribed hereinabove.

In addition, the kit can include a plasmid vector in which a nucleotidesequence encoding a heterologous protein of interest can be subsequentlyinserted for transformation into and expression in a methylotrophicyeast. Alternatively, the knock-in or knock-out vectors in the kits haveconvenient cloning sites for insertion of a nucleotide sequence encodinga heterologous protein of interest.

The kit can also include a methylotrophic yeast strain which can besubsequently transformed with any of the knock-in, knock-out orknock-in-and-knock-out vectors described hereinabove. The kit can alsoinclude a methylotrophic yeast strain which has been transformed withone or more of the knock-in or knock-out vectors. Furthermore, the kitcan include a methylotrophic yeast strain which has been transformedwith a nucleotide sequence encoding and capable of expressing aheterologous glycoprotein of interest.

The present invention is further illustrated by the following examples.

Example 1 Introduction of α-1,2-Mannosidase to the ER-Golgi Border

1.1 Plasmids

Plasmid Promoter Enzyme Tag pGAPZMFManHDEL GAP T. reesei —α-1,2-mannosidase pGAPZMFManMycHDEL GAP T. reesei Myc α-1,2-mannosidasepPICZBMFManMycHDEL AOX1 T. reesei Myc α-1,2-mannosidase pGAPZMFmManHDELGAP mouse mannosidase IB — catalytic domain pGAPZMFmMycManHDEL GAP mousemannosidase IB Myc catalytic domain

The Trichoderma reesei α-1,2-mannosidase gene has been isolated anddescribed by Maras et al. (J. Biotechnol. 77; 255-263, 2000). Thesequence of this gene is available at NCBI Genbank under Accession No.AF212153. A construction fragment was generated by PCR using thepPIC9MFmanase plasmid (same as pPP1MFmds1 described by Maras et al.(2000)) as the template and using the following oligonucleotide primers:5′-GACTGGTTCCAATTGACAAGC-3′ (SEQ ID NO: 2) and5′-AGTCTAGATTACAACTCGTCGTGAGCAAGGTGGCCGCCCCG TCG-3′ (SEQ ID NO: 3). Theresulting product contained the 3′ end of the Pichia pastoris AOXIpromoter, the prepro-signal sequence of the S. cerevisiae α-matingfactor, the open reading frame of the Trichoderma reeseiα-1,2-mannosidase cloned in frame with the signal sequence, the codingsequence for HDEL (SEQ ID NO: 1), a stop codon and an Xba I restrictionsite. This fragment was digested with Eco RI and Xba I, removing the 5′sequences up to the mannosidase ORF, and then cloned into the vectorpGAPZαA (Invitrogen, Baarn, The Netherlands) which had been digestedwith Eco RI and Xba I, thus restoring the fusion with the S. cerevisiaeα-mating factor signal sequence. The resulting plasmid was namedpGAPZMFManHDEL and is graphically depicted in FIG. 1. The ORF sequenceof the MFManHDEL fusion in pGAPZMFManHDEL is set forth in SEQ ID NO: 14.

In order to introduce the coding sequence for a c-Myc tag between thecatalytic domain and the HDEL-signal (SEQ ID NO: 1), the 3′ end of theORF of T. reesei α-1,2-mannosidase was PCR-amplified using a senseprimer 5′-CCATTGAGGACGCATGCCGCGCC-3′ (SEQ ID NO: 4) (containing an Sph Irestriction site) and an antisense primerGTATCTAGATTACAACTCGTCGTGCAGATCCTCTTCTGAGATGAGTTTTTGTTCAGCAAGGTGGCCGCCCCGTCGTGATGATGAA (SEQ ID NO: 5) (containing the codingsequences of the c-Myc tag and the HDEL (SEQ ID NO: 1) signal, followedby a stop codon and an Xba I restriction site). The resulting PCRproduct was digested with Sph I and Xba I, purified by agarose gelelectrophoresis and inserted into pGAPZMFManHDEL which had been cut withthe same restriction enzymes, resulting in plasmid pGAPZMFManMycHDEL. Toput the ORF of pGAPZMFManMycHDEL under the control of the inducible AOXIpromoter, the entire ORF was liberated from pGAPZMFManMycHDEL with BstBI and Xba I, and cloned in pPICZB (Invitrogen, Baarn, The Netherlands),resulting in pPICZBMFManMycHDEL.

Cloning of the mouse mannosidase IB catalytic domain with concomitantaddition of the coding sequence for a C-terminal HDEL-tag (SEQ ID NO: 1)was done by PCR on a mouse cDNA library (mRNA isolated from the L929cell line induced with cycloheximide and mouse Tumor Necrosis Factor.Average insert length of the cDNA library was 2000 bp). The PCRoligonucleotide primers used were: 5′AACTCGAGATGGACTCTTCAAAACACAAACGC3′(SEQ ID NO: 6) and 5′TTGCGGCCGCTTACAACTCGTCGTGTCGGACAGCAGGATTACCTGA3′(SEQ ID NO: 7). The product contained a 5′ Xho I site and the codingsequence for C-terminal HDEL-site, followed by a stop codon and a Not Isite at the 3′ end. The product was cloned in pGAPZαA via the Xho I/NotI sites in the PCR product and the vector, resulting in an in framefusion of the mouse mannosidase catalytic domain with the S. cerevisiaeα-mating factor signal sequence. The sequence of the entire open readingframe generated is set forth in SEQ ID NO: 15.

1.2 Yeast Transformation and Genomic Integration

TABLE 2 Parental strain DNA transformed GS115 (his4) pGAPZMFManHDELpPIC9MFManHDEL pPIC9mManHDEL pPIC9mMycManHDEL pGAPZmManHDELpGAPZmMycManHDEL GS115 (his4 complemented by pGAPZMFManHDELpPIC9InfluenzaHA) pGAPZmManHDEL pGAPZmMycManHDEL PPY120H (his4complemented by pGAPZMFManMycHDEL pPIC9sOCH1) pPICZBMFManMycHDEL yGC4(his4 arg1 ade2 ura3 pPIC9InfluenzaNeuraminidase complemented bypGAPZMFManHDEL pBLURA5′PpOCH1) pPIC9Glucoseoxidase

All transformations to Pichia pastoris were performed withelectroporation according to the directions of Invitrogen. Transformantsof vectors carrying the Zeocin resistance gene were selected on YPDcontaining 100 μg/ml Zeocine (Invitrogen, Baarn, the Netherlands) and 1Msorbitol. Selection of transformants of pPIC9 derivatives was done onminimal medium lacking histidine and containing 1M sorbitol. Genomicintegration of the expression cassettes was verified using PCR ongenomic DNA purified from the Pichia strains using the Yeast Miniprepmethod (Nucleon). In all cases concerning the Trichoderma reesei genefusions, the primers used were the sense primer5′-CCATTGAGGACGCATGCCGCGCC-3′ (SEQ ID NO: 8), which annealed to the 3′half of the mannosidase ORF, and the antisense primer 3′ AOXI5′-GCAAATGGCATTCTGACATCCT-3′ (SEQ ID NO: 9), which annealed to the AOXItranscription terminator that was present in all our expressionconstructs. For the control of genomic integration of the mousemannosidase transgenes, PCR was done using the sense primer 5′GAP5′GTCCCTATTTCAATCAATTGAA3′ (SEQ ID NO: 10, annealing to the GAP promoteror 5′AOXI 5′GACTGGTTCCAATTGACAAGC3′ (SEQ ID NO: 11), annealing to AOXIpromoter), and the antisense primer 3′AOXI (above). For the expressionconstructs containing a Myc tagged Trichoderma reesei α-1,2-mannosidaseexpression unit, further evidence for genomic integration was obtainedusing Southern Blotting with the entire MFManMycHDEL ORF (³²P labelledusing HighPrime, Boehringer Mannheim) as a probe.

1.3 Expression of α-1,2-mannosidase

Expression of an α-1,2-Mannosidase in GS115 strains expressing influenzavirus haemagglutinin was verified by qualitative Northern blot.Expression of an α-1,2-Mannosidase in PPY120H strains was verified byanti-Myc Western blot.

Qualitative Northern Blot—

Total RNA was purified from Pichia strains and the yield was determinedspectrophotometrically. Northern blotting was performed according tostandard procedures and an estimate of the quantity of RNA loaded wasmade using methylene blue staining of the blot, visualizing the rRNAbands. The blot was probed with a ClaI/NarI fragment of the mannosidase,labelled with ³²P using HighPrime (Boehringer Mannheim).

SDS-PAGE and Western Blotting—

Total yeast cell lysates were prepared by washing the cells twice withPBS, followed by boiling in 1 volume of 2× concentrated Laemmli loadingbuffer for 5 min. The lysate was spun briefly in a microcentrifuge priorto gel loading and only the supernatant was loaded. For the analysis ofproteins secreted into the growth media, the proteins were precipitatedfrom 200 μl of these media using desoxycholate/trichloroacetic acidaccording to standard procedures. The pellet was redissolved in 2×concentrated Laemmli loading buffer and the solutions were pH-correctedusing Tris. SDS-PAGE was performed and followed by semidryelectroblotting to nitrocellulose membranes. For Western Blotting, the9E10 anti-Myc and the anti-HA mouse monoclonals (Boehringer Mannheim)were used at a concentration of 1 μg/ml, and the rabbit anti-PDIantiserum (Stressgen) was used at a dilution of 1/500. The secondaryantibodies were goat anti-mouse IgG conjugated to alkaline phosphatasefor the monoclonals and goat anti-rabbit IgG conjugated to peroxidasefor the polyclonal (secondary antibodies from Sigma). Detection wasperformed using the NBT/BCIP system for alkaline phosphatase and theRenaissance substrate (NENBiosciences) for peroxidase. Imaging of thelatter blot result was done on a Lumilager imaging device (BoehringerMannheim).

The results shown in FIG. 4 indicated that the great majority of theHDEL (SEQ ID NO: 1)-tagged protein was retained intracellularly, bothwhen expressed from the strong constitutive GAP promoter and whenexpressed from the strong inducible AOXI promoter.

1.4 Localization of α-1,2-Mannosidase

Isopycnic Sucrose Density Gradient Centrifugation—

To determine the localization of the HDEL (SEQ ID NO: 1)-taggedmannosidase, subcellular fractionation was carried out using cellsexpressing the mannosidase-Myc-HDEL from the strong constitutive GAPpromoter.

Briefly, 0.5 g of wet weight yeast cells were lysed using 4×1 minvortexing with 4.5 g glass beads in 1 ml lysis-buffer (50 mM Tris-HCL pH7.5 containing 0.6 M sorbitol, 10 mM β-mercaptoethanol and 5 mM MgCl₂).Between vortexing periods, the mixture was placed on ice for 5 min. Thesupernatant was collected and the glass beads were washed once withlysis-buffer, and the supernatant of this washing step was added to thefirst supernatant. This lysate was subjected to a differentialcentrifugation procedure. The P10000 pellet was solubilized in 0.5 ml ofa 60% sucrose solution in lysis buffer. This solution was placed at thebottom of an Ultraclear ultracentrifuge tube (Beckman) of 14×89 mm.Subsequently, 1.5 ml each of sucrose solutions of 55, 50, 45, 42.5, 40,and 37.5% were carefully layered over each other. The tube was filled tothe edge with 35% sucrose. Isopycnic sucrose gradient centrifugation wasperformed for 14 h at 180,000 g in a Beckman SW 41 rotor in a BeckmanModel L8-70 preparative ultracentrifuge. After completion, 1 mlfractions were collected from the top and partially dialysed from excesssucrose, evaporated to dryness in a vacuum centrifuge. Afterredissolving the pellet in Laemmli buffer, the samples were subjected toSDS-PAGE in triplicate and the Western blots were treated with anti-HA,anti-Myc or anti-PDI (“PDI” for Protein Disulfide Isomerase),respectively.

The results illustrated almost exact cosedimentation of the MFManMycHDELprotein with the Protein Disulfide Isomerase marker protein (which isalso targeted with a HDEL (SEQ ID NO: 1) signal sequence) (FIG. 5). Inthe same assay, the HA-tagged OCH1 was distributed over the wholegradient, with the highest abundance in fractions having a density lowerthan that of the fractions containing the mannosidase and the PDI. Thisresult indicated that the mannosidase was targeted to the expectedlocation (the ER-Golgi boundary) by the addition of an HDEL (SEQ IDNO: 1) signal. In contrast, the mannosidase without HDEL (SEQ ID NO: 1),expressed from inducible alcohol oxidase I promoter (which was ofcomparable strength as the GAP promoter), was secreted at a high levelof about 20 mg/l.

Immunofluorescence Microscopy—

To confirm the correct targeting of the mannosidase-Myc-HDEL, animmunofluorescence microscopy experiment was performed.

Briefly, yeast cultures were grown to OD₆₀₀ in YPD (forpGAPZMFManMycHDEL) or in YMP following a YPGlycerol growth phase forpPICZBMFManMycHDEL. Formaldehyde was added to the yeast cultures to afinal concentration of 4% and incubated for 10 min at room temperature.Cells were pelleted and resuspended in 50 mM potassium phosphate bufferpH 6.5 containing 1 mM MgCl₂ and 4% formaldehyde and incubated for 2 hat room temperature. After pelleting, the cells were resuspended to anOD₆₀₀=10 in 100 mM potassium phosphate buffer pH 7.5 containing 1 mMMgCl₂ and EDTA-free Complete™ protease inhibitor cocktail (BoehringerMannheim). To 100 μl of cell suspension, 0.6 μl of β-mercapto-ethanoland 200 of 20,000 U/ml Zymolyase 100T (ICN) were added, followed by a 25minute incubation with gentle shaking. The cells were washed twice inthe incubation buffer and added to poly-lysine coated cover slips (theseare prepared using adhesive rings normally in use for reinforcingperforations in paper). Excess liquid was blotted with a cotton swab andthe cells were allowed to dry at 20° C. All blocking, antibodyincubation and washing steps are performed in PBS containing 0.05%bovine serum albumin. Primary antibodies are used at 2 μg/μl andsecondary antibodies conjugated to fluorophores (Molecular probes) wereused at 5 μg/μl. The nucleus was stained with the nucleic acid stainHOECHST 33258. After fixation and cell wall permeabilization, theintegrity of the yeast cell morphology was checked in phase contrastmicroscopy and after immunostaining, the slides were examined under aZeiss Axiophot fluororesensce microscope equipped with a Kodak digitalcamera. Images were processed using Macprobe 4.0 software and preparedwith Corel Photopaint 9.0.

The Golgi marker protein OCH1-HA gave the typical Golgi staining patterndescribed in the literature (speckle-like staining). Staining with the9E10 monoclonal anti-Myc antibody, recognizing mannosidase-Myc-HDEL,gave a perinuclear staining pattern with some disparate staining in thecytoplasm, highly indicative for an ER targeting (FIG. 4).

Based on the foregoing experiments, it is concluded that the Trichodermareesei mannosidase-Myc-HDEL was targeted to the ER-Golgi boundary.

Example 2 Co-Expression of Mannosidase-HDEL with RecombinantGlycoproteins

Co-expression of Mannosidase-HDEL with the Trypanosoma cruzitrans-Sialidase

The cloning of a Trypanosoma cruzi trans-sialidase gene coding for anactive trans-sialidase member without the C-terminal repeat domain hasbeen described by Laroy et al. (Protein Expression and Purification 20:389, 2000) which is incorporated herein by reference. The sequence ofthis Trypanosoma cruzi trans-sialidase gene is available through NCBIGenbank under the Accession No. AJ276679. For expression in P. pastoris,the entire gene was cloned in pHILD2 (Invitrogen, San Diego, Calif.),creating pHILD2-TS. To allow better secretion, pPIC9-TS was created inwhich trans-sialidase was linked to the prepro secretion signal of theyeast α-mating factor. Plasmids pPIC9-TSE and pCAGGS-prepro-TSE werecreated where the epitope E-tag was added to the C-terminal of thetrans-sialidase to allow easy detection and purification. Theconstruction of pHILD2-TS, pPIC9-TSE and pCAGGS-prepro-TSE has beendescribed by Laroy et al. (2000), incorporated herein by reference. Thevectors used in the construction were made available through for pCAGGS(No. LMBP 2453), Invitrogen, San Diego, Calif. for pHILD2 and pPIC9, andPharmacia Biotech for pCANTAB-5E.

Plasmid pPIC9-TSE was linearized with SstI and was transformed into P.P. pastoris GS115 (his4) strain by electroporation according to themanufacturer's instructions (Invitrogen). One of the transformants wasfurther transformed with plasmid pGAPZMFManHDEL, establishing a strainco-expressing Mannosidase-HDEL and the Trypanosoma cruzitrans-sialidase.

Fermentation and protein purification was according to the proceduresdescribed by Laroy et al. (2000).

Purified trans-sialidase was subject to carbohydrate analysis accordingto Callewaert et al., Glycobiology 11, 4, 275-281, 2001. Briefly, theglycoproteins were bound to the PVDF membrane in the wells of a 96-wellplate, reduced, alkylated and submitted to peptide-N-glycosidase Fdeglycosylation. The glycans were derivatised with8-amino-1,3,6-pyrenetrisulfonic acid by reductive amination.Subsequently, the excess free label was removed using SephadexG10-packed spin columns and the glycans were analysed by electrophoresison a 36 cm sequencing gel on an ABI 377A DNA-sequencer and detectedusing the built-in argon laser. Digests with 3 mU/ml purified T. reeseiα-1,2-mannosidase (described by Maras et al., J. Biotechnol. 77, 255-63,2000) were also performed in 20 mM sodium acetate pH=5.0. The glycansderived from 1 μg of the purified recombinant glycoproteins were used asthe substrate. 1 U of the α-1,2-mannosidase is defined as the amount ofenzyme that releases 1 μmol of mannose from baker's yeast mannan perminute at 37° C. and pH=5.0.

As can be seen in FIG. 6, panel B, the major N-glycan on trans-sialidasewas Man₈GlcNAc₂ (Compare with panel F, representing an analysis of theN-glycans of bovine RNAseB. The one but last peak in this profile isMan₈GlcNAc₂, the first peak is Man₅GlcNAc₂). In vitro, this glycan wasdigestible to Man₅GlcNAc₂ with α-1,2-mannosidase (FIG. 6, panel C). Inthe N-glycan profile of the trans-sialidase co-expressed withmannosidase-HDEL, the major peak corresponded to Man₅GlcNAc₂ (FIG. 6,panel D).

Co-Expression of Mannosidase-HDEL with the Influenza a VirusHaemagglutinin

The Influenza A virus haemagglutinin was known to be glycosylated inPichia pastoris with high-mannose N-glycans containing 9-12 mannoseresidues (Saelens et al. Eur. J. Biochem. 260: 166-175, 1999). Theeffect of a co-expressed mannosidase on the N-glycans of thehaemagglutinin was assessed in an N-glycan profiling method describedbelow. In addition, to compare the efficiency of the Trichoderma enzyme(having a temperature optimum of 60° C.) with a mammalian mannosidasehaving a temperature optimum of 37° C., the catalytic domain of themouse mannosidase IB from a mouse cDNA-library was cloned and taggedwith a HDEL signal by PCR amplification. This ORF was cloned after theprepro-signal sequence of the S. cerevisiae α-mating factor under thecontrol of the GAP promoter. Expression of the mannosidase-HDELtransgenes on the mRNA level was confirmed by qualitative Northernblotting.

The haemagglutinin was expressed and purified from a non-mannosidaseexpressing control strain and from a strains co-expressing theTrichoderma reesei mannosidase-HDEL or the mouse mannosidase IB-HDELaccording to the procedure described by Kulakosky et al. Glycobiology 8:741-745 (1998). The purified haemagglutin was subjected to PNGase Fdigestion as described by Saelens et al. Eur. J. Biochem. 260: 166-175,1999. The proteins and glycans were precipitated with 3 volumes ofice-cold acetone and the glycans were extracted from the pellet with 60%methanol. Following vacuum evaporation, the glycans were labeled with8-amino-1,3,6 pyrenetrisulfonic acid by adding 1 μl of a 1:1 mixture of20 mM APTS in 1.2M citric acid and 1M N_(a)CNBH₃ in DMSO and incubatingfor 16 h at 37° C. at the bottom of a 250 μl PCR-tube. The reaction wasstopped by the addition of 10 μl deionized water and the mixture wasloaded on a 1.2 cm Sephadex G10 bed packed to dryness in amicrospin-column by centrifugation in a swinging bucket rotor, whichprovided for a flat resin surface. After loading, 50 μl deionised waterwas carefully added to the resin bed and the spin column was brieflycentrifuged for 5 seconds at 750 g in a tabletop centrifuge. Thiselution process was repeated twice and all the eluates were pooled andevaporated to dryness in a Speedvac vacuum centrifuge (Savant). Thelabeled glycans were reconstituted in 1.5 μl gel loading buffercontaining 50% formamide and 0.5 μl Genescan 500™, labeled withrhodamine (Perkin Elmer Bioscience), serving as an internal referencestandard. This mixture was loaded on a DNA-sequencing gel containing 10%of a 19:1 mixture of acrylamide:bisacrylamide (Biorad, Hercules, Calif.,USA) and made up in the standard DNA-sequencing buffer (89 mM Tris, 89mM borate, 2.2 mM EDTA). Polymerization of the gel was catalyzed by theaddition of 200 μl 10% ammononiumpersulfate solution in water and 20 μlTEMED. The gel was of the standard 36 cm well-to-read length and was runon an Applied Biosystems Model 373A DNA-sequencing apparatus. Prerunningof the gel was done at 1000 V for 15 min. and after loading, the gel waselectrophoresed for 8 h at 1250 V without heating. This methodologygives a limit of detection of 10 fmol per peak. The data were analysedwith Genescan 3.0 software.

As shown in FIG. 7, the Trichoderma reesei α-1,2-mannosidase providedthe most complete reduction in the number of α-1,2-mannoses present onthe N-glycans. The N-glycan processing by mouse mannosidase IB-HDEL wasless efficient than by the Trichoderma reesei α-1,2-mannosidase.

Despite the efficient removal of α-1,2-mannoses from the N-glycans ofhaemagglutinin, no Man₅GlcNAc₂ was obtained. Even after digestion of theN-glycans with 3 mU of purified Trichoderma reesei α-1,2-mannosidase,only Man₆GlcNAc₂ was obtained as the smallest sugar chain. These resultsindicated that the remaining residues were possibly α-1,6-linkedmannoses, originating from the initiating OCH1 α-1,6-mannosyltransferaseenzymatic activities. OCH1 was observed to be localized to very earlypart of the Golgi apparatus and could act on the N-glycans ofhaemagglutinin before complete digestion of the Man₈GlcNAc₂ precursor toMan₅GlcNAc₂ by the mannosidases-HDEL. Thus, for proteins whose glycansare efficiently modified by the α-1,6-mannosyltransferase, aninactivation of the OCH1 gene coding for the transferase would bedesirable in order to obtain proteins with Man₅GlcNAc₂.

Example 3 Inactivation of the Pichia Och1 Gene

A Pichia pastoris sequence was found in the GenBank under Accession No.E12456 and was described in Japanese Patent Application No. 07145005,incorporated herein by reference. This sequence shows all typicalfeatures of an α-1,6-mannosyltransferase and is most homologous to theS. cerevisiae OCH1, thus referred to herein as the Pichia pastoris Och1gene.

First, the full ORF of the Pichia pastoris Och1 gene was PCR cloned inpUC18 to obtain plasmid pUC18pOch1. pUC18pOch1 was cut with HindIII,blunt-ended with T4 polymerase, then cut with XbaI, releasing a fragmentcontaining the 5′ part of the Pichia pastoris Och1 gene. This fragmentwas ligated into the vector pBLURA IX (available from the Keck GraduateInstitute, Dr. James Cregg, which had been cut with Eco RI, blunt-endedwith T4 polymerase, and then cut with Nhe I. This ligation generatedpBLURA5′PpPCH1, as shown in FIG. 8.

Disruption of this Pichia OCH1 gene in the Pichia genome was achieved bysingle homologous recombination using pBLURA5′PpOCH1, as illustrated inFIG. 9. As a result of the single homologous recombination, the Och1gene on the Pichia chromosome was replaced with two Och1 sequences: oneconsisted only about the first one third of the full Och1 ORF, the otherhad a full Och1 ORF without a Och1 promoter. Single homologousrecombination was achieved as follows. Cells of the Pichia strain yGC4were transformed by electroporation with pBLURA5′PpOCH1 which had beenlinearized with the single cutter Bst BI. About 500 transformants wereobtained on minimal medium containing 1M sorbitol, biotin, arginine,adenine and histidine and incubation at 27° C. Thirty-two of thesetransformants were picked and re-selected under the same conditions.Twelve clones were further analyzed for correct genomic integration ofthe cassette by PCR. Seven of the twelve URA prototrophic clonescontained the cassette in the correct locus.

One of the Och1-inactivated clones was also further transformed withpGAPZMFManHDEL to produce “supertransformants”. Both theOch1-inactivated clone and three supertransformants also expressing theManHDEL were evaluated in cell wall glycan analysis as follows. Yeastcells were grown in 10 ml YPD to an OD₆₀₀=2 and mannoproteins wereprepared by autoclaving the yeast cells in 20 mM sodium citrate bufferpH7 for 90 min at 120° C. and recovery of the supernatant aftercentrifugation. Proteins were precipitated from this supernatant with 3volumes of cold methanol. The protein preparation obtained in this waywas used for N-glycan analysis using DSA-FACE as described by Callewaertet al. (2001) Glycobiology 11, 275-281. As shown in FIG. 10, there wasan increased amount of Man₈GlcNAc₂ glycan in the Och1-inactivanted cloneas compared to parent strain yGC4, indicative of a reduced activity ofthe Och1 enzyme. In all three supertransformants which also expressedthe HDEL (SEQ ID NO: 1)-tagged α-1,2 mannosidase, the production ofMan₅GlcNAc₂ was observed. Furthermore, upon digestion of the same glycanmixtures with 3 mU/ml purified recombinant Trichoderma reeseiα-1,2-mannosidase, more Man₅GlcNAc₂ was formed in the strain transformedwith pBLURA5′PpOCH1 than in the parent strain (FIG. 11, compare panel 2and 3).

These results confirmed that the lack of a production of Man₅ glycans onrecombinantly produced proteins such as haemagglutinin from cellsexpressing α-1,2-mannosidase were due to the activity of the Och1protein. These results further indicate that the production ofglycoproteins with Man₅ glycans could be facilitated by the inactivationof the Och1 gene.

Example 4 Expression of Glucosidase II in Pichia pastoris

4.1 Amplification of the GLSII Alpha Subunit ORF from S. cerevisiae.

Genomic DNA was prepared from the S. cerevisiae strain INVS (α, leu2-3,112 h is 3Δ1, trp1-289, ura3-52), using the Nucleon kit (Amersham). Atouch-down PCR reaction was performed using this genomic DNA as templateand the LA TaKaRa polymerase (ImTec Diagnostics). The sequence of thePCR primers was based on the known sequence of the S. cerevisiae GLSIIORF:

Sense primer: (SEQ ID NO: 12) 5′ CCG CTC GAG ATG GTC CTT TTG AAA TGG CTC3′           Xho I Antisense primer: (SEQ ID NO: 13) 5′ CCG GGC CCA AAAATA ACT TCC CAA TCT TCA G 3′        Apa I4.2 Cloning of the S. cerevisiae Glucosidase II ORF into Pichia pastorisExpression Vectors.

Construction of the Glucosidase II Expression Vectors—

The PCR fragment was digested with Xho I/Apa I and ligated into thepGAPZA vector (Invitrogen), thereby placing the ORF under thetranscriptional control of the GAP promoter. Using this strategy, themyc and the His6 tag were placed in frame to the C-terminus ofGlucosidase II, creating pGAPZAGLSII. The complete ORF of pGAPZAGLSIIwas then sequenced to ensure that no mutations were generated in the PCRreaction. The sequence of the vector pGAPZAGLSII was set forth in SEQ IDNO: 18. The GLSII ORF from the pGAPZAGLSII vector was cloned into vectorpPICZA (Invitrogen) to create pPICZAGLSII, thereby placing the ORF underthe transcriptional control of the AOXI promoter. The GLSII ORF from thepGAPZAGLSII vector was cloned into vector pAOX2ZA, thereby placing theORF under the transcriptional control of the AOX2 promoter. This vectorwas created by replacing the multi cloning site of vector pAOX2ZB withthe multi cloning site of pPICZA. Vector pAOX2ZB was generated byreplacing the AOX1 promotor of pPICZB by the AOX2 promotor region of theAOX2 gene (Martinet et al., Biotechnology Letters 21). The AOX2 promotorregion was generated by PCR on Pichia genomic DNA with the sense primer5′GACGAGATCTTTTTTTCAGACCATATGACCGG 3′ (SEQ ID NO: 26) and the antisenseprimer 5′GCGGAATTCTTTTCTCAGTTGATTTGTTTGT 3′ (SEQ ID NO: 27). The GLSIIORF from the pGAPZGLSII vector was cloned into vector pYPT1ZA to createpYPTIZAGLSII, thereby placing the ORF under the transcriptional controlof the YPT1 promoter. Vector pYPTZA was created by replacing the AOX1promoter of pPICZA by the YPT1 promoter present on the plasmid pIB3(GenBank accession number AF027960)(Sears et al., Yeast 14, pg 783-790,1998). All constructs contain the phleomycin resistance gene. Theresulting final expression vectors (pGAPZAGLSII, pAOX2ZAGLSII,pPICZAGLSII and pYPT1ZAGLSII) are depicted in FIGS. 12-15.

Similar expression vectors were constructed, carrying the Ampicillinresistance marker and the Pichia ADE1 selection marker. In principle,the Zeocin resistance expression cassette of the plasmids pAOX2ZAGLSII,pGAPZAGLSII and pYPT1ZAGLSII was replaced by the Ampicillin and PichiaADE1 cassette of the vector pBLADE IX (Cregg, J. M.) to result in thevectors pAOX2ADE1glsII, pGAPADE1glsII and pYPT1ADE1glsII. VectorpPICADE1glsII was obtained by inserting the glucosidase II open readingframe into the multiple cloning site of the vector pBLADE IX (Cregg, J.M.). The resulting final expression vectors (pGAPADE1glsII,pAOX2ADE1glsII, pPICADE1glsII and pYPT1ADE1glsII) are depicted in FIGS.16-20.

Adding the ER Retention Tag HDEL to Glucosidase II Expression Vectors—

The following primers were used to generate an HDEL-containing PCRfragment:

Primer 1: (SEQ ID NO: 28) 5′GCG GGT CGA C/ CA C/GA C/GA A/CT G/TG A/GTTTT          Sal I   H    D    E    L    stop AGC CTT AGA CAT GAC 3′Primer 2: (SEQ ID NO: 29) 5′CAG GAG CAAA GCT CGT ACG AG 3′                     Spl I

PCR was performed on pGAPZAGLSII with Taq pol., at 60° C. The PCRfragment of 225 bp was cut with Sal I/Spl I and ligated into the SalI/Spl I opened pGAPZAGLSII vector, creating plasmid pGAPZAglsIIHDEL. Thesequence of plasmid pGAPZAglsIIHDEL is set forth in SEQ ID NO: 24. Theconstruction strategy and the resulting final expression vectors(pGAPZAglsIIHDEL and pGAPADE1glsIIHDEL) are depicted in FIGS. 20-21.

4.3 Transformation of a Pichia pastoris Strain.

Transformation was performed using the conventional electroporationtechniques, as described by Invitrogen. Cells of the Pichia pastorisstrain PPY12-OH were transformed with pGAPZGLSII which had been cut withthe single cutter Avr II. Transformants were selected based on theirresistance to zeocin.

Genomic Analysis of the Transformants—

Genomic DNA was prepared from some zeocin resistant Pichiatransformants. A PCR reaction was performed on the genomic DNA in orderto determine whether or not the glucosidase II gene was integrated intothe yeast genome. PCR was performed using Taq DNA polymerase (Boehinger)(2.5 mM MgCl₂, 55° C. for annealing). The primers were the same as theones we used for the amplification of the ORF on S. cerevisiae genomicDNA. pGAPZAGLSII transformants were confirmed by the presence of aspecific PCR product indicative of the glucosidase II ORF.

4.4 Expression and Secretion of the S. Cerevisiae Glucosidase II AlphaSubunit in Pichia pastoris

Analysis at the Transcriptional Level—

RNA was prepared from the transformants which scored positive after thegenomic analysis. RNA was prepared using acid phenol. From each sample,15 μg of RNA was loaded on a formaldehyde agarose gel. Afterelectrophoresis the RNA was blotted on a Hybond N membrane. The membranewas hybridizing using a radioactive probe, which consists of a 344 bpglucosidase II specific fragment, corresponding to the 3′ region of theglucosidase II ORF. No signals were detected with non-transformedcontrol strains, whereas clear signals were observed with transformants.

Analysis at the Protein Level Using a Double Membrane Assay—

A nitrocellulose membrane was placed on a buffered dextrose medium(BMDY). On top of that nitrocellulose membrane, a cellulose acetatemembrane was placed. Pichia transformants of pGAPZAGLSII were streakedon the cellulose acetate and grown for a few days. The yeast cellsremained on the cellulose acetate, while the secreted proteins crossedthis membrane. As such the secreted protein was captured onto thenitrocellulose membrane. After a few days the cellulose acetate,containing the yeast colonies, was removed. The nitrocellulose membranewas analyzed for the presence of glucosidase II using anti-myc antibody.Most of the transformants gave a clear signal as compared to a faint,hardly visible signal with the WT, non-transformed strain.

Extracellular Expression—

PPY12-OH transformants of the construct pGAPZAGLSII(mychis6) (strains12, 14 and 18) and transformants of the construct pGAPZAGLSII(myc)HDEL(strains H1, H2 and H3) were grown for 2 days on 2×10 ml BMDY medium.These 6 transformants earlier scored positive both on the genomic level(PCR on gDNA) and on the RNA level (Northern blot). The culture mediumwas collected by centrifugation and concentrated with Vivaspin columnsto about 1 ml. Proteins from this concentrate were precipitated withTCA, resuspended in Laemmli buffer and loaded for SDS-PAGE analysis.Proteins were blotted to nitrocellulose membrane. The blot was incubatedovernight with anti-myc Ab. The secondary Ab was linked to peroxidase.Using the Renaissance luminiscence detection kit (NEN) and a lightsensitive film (Kodak), a strong band at about 110 kDa was observed forthe transformants 12, 14 and 18, indicating that GLSII was expressed andsecreted from these transformants. No signal was obtained for thetransformants H1-3, which indicate that the HDEL (SEQ ID NO: 1) tag,which was added C-terminally to the GLSII ORF, resulted in an ERlocalization of the protein, preventing GLSII to be secreted into thegrowth medium.

Intracellular Expression—

The 6 transformants and the WT strain were grown for 2 days in 500 mlBMDY. The cells were collected by centrifugation, washed, resuspendedinto a minimal volume (50 mM Tris.HCl pH 7.5, 5% glycerol) and brokenusing glass beads. The cell debris was removed through severalcentrifugation steps (low speed centrifugation (2000-3000 g)). Membraneswere obtained from the supernatant through ultracentrifugation. Thepellets were resuspended in Laemmli buffer and loaded for SDS-PAGEanalysis. The proteins were blotted on a nitrocellulose membrane. Theintracellular GLSII expression was checked using anti-myc Ab andperoxidase conjugated secondary Ab. Following the luminescencedetection, a band at about 110 kDA was observed with the GLSIIHDELtranformants (H1 and H3, faint signal for H2), but not with the WT andGLSII expression strains. These results clearly indicate theintracellular presence of the recombinant GLSII when expressed with aC-terminal HDEL (SEQ ID NO: 1) tag. No GLSII was detectedintracellularly when this tag was not present.

4.5 Purification and Activity Assays of the Recombinant Glucosidase IIAlpha Submit

A GLSII assay was set up as follows and was tested using a commerciallyavailable yeast alpha-glucosidase (Sigma) as a positive control.

Composition: 70 μl 80 mM phosphate-citrate buffer pH 6.8, 7 μl 250 mMmannose, 3.5 μl 250 mM 2-deoxy-D-glucose, 0.8 μl4-MeUmbelliferyl-alpha-D-glucopyranoside (1 μM). Three assays wereperformed: one with 1 unit commercial enzyme, one without the enzyme andone with the enzyme but without the substrate. The assay mixture wasincubated overnight at 30° C. When illuminated with UV, only thereaction mixture with both the enzyme and the substrate showedfluorescence (FIG. 22). This indicates that the assay was very specificin detecting the activity of the alpha-glucosidase.

WT PPY12-OH, strain 18 and strain H3 were grown during 2 days in 2×10 mlgrowth medium. Cells were spun down and medium was adjusted to 300 mMNaCl and 10 mM imidazol and concentrated with Vivaspin columns to 0.5-1ml. Medium was loaded onto a Ni-NTA spin column (Qiagen) and thepurification was performed according to the manufacturesrecommendations. Protein was eluted from the column in 2×100 μl elutionbuffer (50 mM NaH₂PO₄, 300 mM NaCl, 250 mM imidazol pH 8.0). From eacheluate, 20 μl was assayed for its glucosidase II activity. 0.33 units ofthe commercial enzyme diluted in 20 μl of the elution buffer was used asa positive control. Fluorescence was observed with the positive controland the elute of strain 18, the strain which secreted the enzyme intothe growth medium. These results indicate that the recombinant S.cerevisiae GLSII alpha subunit, secreted by Pichia pastoris, was afunctionally active enzyme. The activity was not seen in the WT(untransformed) strain, nor in strain H3 as the GLSII was expressedintracellularly (FIG. 23). These results also indicate that the betasubunit is not necessary for the functionality of the alpha part of theprotein.

Example 5 Creating Pichia Strains Expressing Both Glucosidase II andMannosidase

Strain GS 115 was transformed with pGAPZGLSII and pGAPZglsIIHDEL.Transformants were selected on YPDSzeo.

Strain yGC4 was transformed with the following constructs, respectively:

(1) pGAPADEglsII and pGAPADEglsIIHDEL, selection on synthetic sorbitolmedium without adenine;

(2) pGAPZMFManHDEL: selection on YPDSzeo; and

(3) pGAPZMFManHDEL/pGAPADEglsIIHDEL: selection on synthetic sorbitolmedium without adenine and with zeocin.

Strain yGC4 with OCH1 knock-in and expressing MFmannosidaseHDEL wastransformed with pGAPADEglsII and pGAPADEglsIIHDEL. Selection oftransformants was done on synthetic sorbitol medium without adenine anduracil.

For all transformations, colonies were obtained. Transformants with theexpression vector(s) integrated into the genome, determined by PCR, wereobtained. Expression of GLSII from some of these transformants wasobserved.

The invention claimed is:
 1. A genetically engineered Pichia strain, which lacks a functional enzyme involved in production of high mannose structures, and comprises an exogenous nucleic acid encoding and expressing an exogenous enzyme for production of Man₅GlcNAc₂, wherein said enzyme involved in production of high mannose structures is α-1,6-mannosyltransferase encoded by the OCH1 gene, said exogenous enzyme for production of Man₅GlcNAc₂ is α-1,2-mannosidase or an enzymatically active fragment thereof and is targeted to the endoplasmic reticulum (ER), wherein said OCH1 gene is disrupted in said strain and the OCH1 gene disruption is the sole genetic disruption of genes coding for Golgi mannosyl transferases acting in N-glycosylation of said strain, and wherein said strain produces Man₅GlcNAc₂ as a N-glycan structure or an intermediate N-glycan structure.
 2. The strain of claim 1, wherein said exogenous enzyme is of a fungal origin or a mammalian origin.
 3. The strain of claim 1, wherein the targeting of said exogenous enzyme to the ER is achieved by engineering said exogenous enzyme to include the ER retention signal as set forth in SEQ ID NO:
 1. 4. A method for producing a glycoprotein with reduced hyperglycosylation in Pichia, comprising: (i) providing a Pichia strain, which lacks a functional enzyme involved in production of high mannose structures, and comprises an exogenous nucleic acid encoding and expressing an exogenous enzyme for production of Man₅GlcNAc₂, wherein said enzyme involved in production of high mannose structures is α-1,6-mannosyltransferase encoded by the OCH1 gene, said exogenous enzyme for production of Man₅GlcNAc₂ is α-1,2-mannosidase or an enzymatically active fragment thereof and is targeted to the ER, wherein said OCH1 gene is disrupted in said strain and the OCH1 gene disruption is the sole genetic disruption of genes coding for Golgi mannosyl transferases acting in N-glycosylation of said strain, and wherein said strain produces Man₅GlcNAc₂ as a N-glycan structure or an intermediate N-glycan structure; and (ii) producing said glycoprotein in said Pichia strain.
 5. The method of claim 4, wherein said exogenous enzyme is of a fungal origin or a mammalian origin.
 6. The method of claim 4, wherein the targeting of said exogenous enzyme to the ER is achieved by engineering said exogenous enzyme to include the ER retention signal as set forth in SEQ ID NO:
 1. 7. The strain of claim 1, wherein said α-1,2-mannosidase is T. reesei α-1,2-mannosidase, and said strain produces Man₅GlcNAc₂ as a predominant N-glycan structure or a predominant intermediate N-glycan structure.
 8. The method of claim 4, wherein said α-1,2-mannosidase is T. reesei α-1,2-mannosidase, and said strain produces Man₅GlcNAc₂ as a predominant N-glycan structure or a predominant intermediate N-glycan structure. 